Computational Analysis of the Inhibition Mechanism of NOTUM by the ONIOM Method

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Notum, a serine hydrolase, regulates the Wingless-related integration site (Wnt) signaling pathway by cleaving palmitoleate from Wnt ligands.
• Dysregulation of Wnt signaling is implicated in diseases such as osteoporosis, cancer, and Alzheimer's disease.
• Irreversible inhibitors targeting Notum are being developed, with recent advancements in 4-(indolin-1-yl)-4-oxobutanoic acid esters.

Purpose of the Study:

• To computationally investigate the irreversible inhibition mechanism of Notum by 4-(indolin-1-yl)-4-oxobutanoic acid esters.
• To elucidate the roles of active-site residues in the enzyme-inhibitor interaction and adduct hydrolysis.
• To provide insights into both the inhibition and catalytic mechanisms of Notum.

Main Methods:

• Utilized the n-layered integrated molecular orbital and molecular mechanics (ONIOM) method for computational modeling.
• Employed density functional theory (DFT) within the ONIOM framework to analyze reaction energetics.
• Modeled transesterification between the catalytic serine (Ser-232) and the inhibitor, followed by hydrolysis of the enzyme-inhibitor adduct.

Main Results:

• The hydrolysis of the covalent enzyme-inhibitor adduct was found to be highly endergonic.
• This high energy barrier supports the proposed mechanism of irreversible inhibition.
• Key active-site residues were identified as playing significant roles in the inhibition process.

Conclusions:

• The computational study confirms the irreversible nature of Notum inhibition by the studied compounds.
• The findings offer a deeper understanding of Notum's catalytic and inhibition mechanisms.
• This knowledge can guide the design of more effective therapeutic agents targeting Notum for various diseases.